Flavokawain B targets protein neddylation for enhancing the anti-prostate cancer effect of Bortezomib via Skp2 degradation

Xuesen Li, Victor Pham, Matthew Tippin, Dongjun Fu, Raymond Rendon, Liankun Song, Edward Uchio, Bang H. Hoang, Xiaolin Zi

Research output: Contribution to journalArticle

Abstract

Background: Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown. Methods: An in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods. Results: FKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1-452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn't attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2. Conclusion: These findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.

Original languageEnglish (US)
Article number25
JournalCell Communication and Signaling
Volume17
Issue number1
DOIs
StatePublished - Mar 18 2019
Externally publishedYes

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Prostatic Neoplasms
Degradation
Proteins
Assays
Cells
Growth
Bortezomib
flavokawain B
Cell Line
Down-Regulation
Hot Temperature
Kava
Western Blotting
Apoptosis
Proteasome Inhibitors
Ubiquitin-Protein Ligases
Castration
Cell growth
Proteasome Endopeptidase Complex
Enzymes

Keywords

  • And prostate cancer
  • Chalcone
  • Neddylation
  • Skp2

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Flavokawain B targets protein neddylation for enhancing the anti-prostate cancer effect of Bortezomib via Skp2 degradation. / Li, Xuesen; Pham, Victor; Tippin, Matthew; Fu, Dongjun; Rendon, Raymond; Song, Liankun; Uchio, Edward; Hoang, Bang H.; Zi, Xiaolin.

In: Cell Communication and Signaling, Vol. 17, No. 1, 25, 18.03.2019.

Research output: Contribution to journalArticle

Li, Xuesen ; Pham, Victor ; Tippin, Matthew ; Fu, Dongjun ; Rendon, Raymond ; Song, Liankun ; Uchio, Edward ; Hoang, Bang H. ; Zi, Xiaolin. / Flavokawain B targets protein neddylation for enhancing the anti-prostate cancer effect of Bortezomib via Skp2 degradation. In: Cell Communication and Signaling. 2019 ; Vol. 17, No. 1.
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T1 - Flavokawain B targets protein neddylation for enhancing the anti-prostate cancer effect of Bortezomib via Skp2 degradation

AU - Li, Xuesen

AU - Pham, Victor

AU - Tippin, Matthew

AU - Fu, Dongjun

AU - Rendon, Raymond

AU - Song, Liankun

AU - Uchio, Edward

AU - Hoang, Bang H.

AU - Zi, Xiaolin

PY - 2019/3/18

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N2 - Background: Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown. Methods: An in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods. Results: FKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1-452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn't attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2. Conclusion: These findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.

AB - Background: Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown. Methods: An in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods. Results: FKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1-452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn't attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2. Conclusion: These findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.

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KW - Chalcone

KW - Neddylation

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